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Isolation of Mutants Defective in -Amylase from Bacillus subtilis: Genetic Analyses

^a Division of Enzymology, The Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

ABSTRACT

The rate of -amylase (EC 3.2.1.1) synthesis in Bacillus subtilis is regulated by a gene, amyR, located near a structural gene, amyE, for the enzyme. To construct a fine map of the amyR-amyE region, we isolated 28 mutants defective in -amylase activity. Eleven mutants out of 28 showed no -amylase activity, whereas the other 17 showed less -amylase activity than the parent. Out of 17 partially positive -amylase mutants, 10 produced temperature-sensitive enzymes, and 4 produced immunologically altered enzymes, two of which are concurrently temperature-sensitive, and 5 produced smaller amounts of -amylases which are indistinguishable from normal enzyme in their temperature sensitivity and immunological properties. Two out of 11 -amylase-negative mutants produced material that cross-reacted with anti-amylase serum, and 3 mutants carried suppressible mutations by the suppressor described by Okubo. Mapping data indicate that all 28 mutation sites are located in the amyE region, and none of the groups of the mutants mentioned above contains lesions that are clustered in a single region of amyE. The amyR gene seems most likely to adjoin the terminal region of amyE.

¹ Present address: Biophysics Division, Cancer Research Institute, Kanazawa University, 13-1, Takara-machi, Kanazawa, Japan.